Method of treating lysosomal storage disorders

ABSTRACT

Therapeutic compositions and methods for treatment of late-onset Gaucher disease are described herein. The compositions comprise compounds having activity as pharmacological chaperones for mutant forms of the beta-glucocerebrosidase. Methods of treatment involve providing therapeutically effective amounts of such compositions to subjects in need thereof.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a continuation of co-pending U.S. patent application Ser. No.13/676,506, filed Nov. 14, 2012, which is a divisional of U.S. patentapplication Ser. No. 13/248,434, filed Sep. 29, 2011, now U.S. Pat. No.8,404,668, which is a divisional of U.S. patent application Ser. No.12/229,445, filed Aug. 22, 2008, now U.S. Pat. No. 8,124,597, the entiredisclosure of which is incorporated herein by reference, which claimsthe filing date benefit of the following U.S. Provisional Applications,the entire disclosures of which are incorporated herein by reference:No. 60/972,968, filed Sep. 17, 2007; No. 61/065,550, filed Feb. 12,2008, and No. 61/065,684, filed Feb. 13, 2008.

FIELD OF THE INVENTION

The invention relates to the use of small molecule compounds to treatGaucher Disease.

BACKGROUND OF THE INVENTION

Lysosomal storage disorders (LSD's) are a group of diseases resultingfrom the abnormal metabolism of various substrates, includingglycosphingolipids, glycogen, mucopolysaccharides and glycoproteins. Themetabolism of exo- and endogenous high molecular weight compoundsnormally occurs in the lysosomes, and the process is normally regulatedin a stepwise process by degradation enzymes. For example, the enzymesbeta-glucocerebrosidase and alpha-galactosidase are involved in thecatabolism of glycosphingolipids.

A deficient activity in even one catabolic enzyme may impair theprocess, resulting in an accumulation of particular substrates. As partof lysosomal processing, the enzyme beta-glucocerebrosidase (referred tointerchangeably herein as “GCase” or “Gcc”) cleaves the terminal glucoseresidue from glucosylceramide. A deficiency in GCase—in the form of oneor more amino acid mutations—results in the LSD known as Gaucherdisease. In Gaucher disease, various forms of mutant GCase have reduced,little, or no glucosylceramide cleavage activity, depending upon themutated amino acid or amino acids. The severity of this disorder iscorrelated with relative levels of residual enzyme activity and theresulting extent of accumulation of the substrate.

Currently, Gaucher patients are treated using an expensive enzymereplacement therapy at a cost of about $300,000 USD per year perpatient, or by using non-specific substrate reduction therapy. Withthese treatments, the enzyme deficiency itself is not treated, butrather, the accumulation of substrate is treated by reducing thesynthetic levels of all gangliosides.

What is needed is an efficient and less-expensive way to treatindividuals having Gaucher disease. The present invention addresses andmeets this need.

Bromhexine, and its metabolite ambroxol, are expectoration improvers andmucolytic agents used in the treatment of respiratory disordersassociated with viscid or excessive mucus. They work to decrease mucusviscosity by altering its structure. Neither bromhexine or ambroxol havebeen known to have any activity relevant to the treatment of lysosomalstorage disorders.

SUMMARY OF THE INVENTION

The invention relates to a method of treating an individual sufferingfrom Gaucher disease, comprising administering to the individual aneffective amount of ambroxol, or a derivative thereof. The inventionalso relates to ambroxol, or a derivative thereof, as a medicament fortreatment of Gaucher disease. The invention also relates to acomposition for the treatment of Gaucher disease comprising atherapeutically effective amount of ambroxol, or a derivative thereof,together with a pharmaceutically acceptable excipient. The inventionalso comprises a method of dosing ambroxol, or derivative thereof, toachieve optimal pharmacological chaperoning and enzyme enhancement.

In some embodiments, the ambroxol derivative comprises a cyclohexanering, an amine group, and/or at least two bromine groups. In someembodiments, the derivative comprises a compound listed in Table 2. Insome embodiments, the derivate is an enantiomer, analog, ester, amide,prodrug, or metabolite of ambroxol, or a salt of ambroxol, particularlya pharmaceutically acceptable salt. In some embodiments, the ambroxolderivative is bromhexine or a salt of bromhexine. In some embodiments,the salt of ambroxol or bromhexine is a hydrochloride.

In an embodiment of the treatment method of the invention, theindividual also suffers from Parkinson's disease.

The invention also relates to a method of treating an individualsuffering from Gaucher disease, comprising administering to theindividual an effective amount of a compound listed in Table 1. Theinvention also relates to a compound listed in Table 1, as a medicamentfor treatment of Gaucher disease. The invention also relates to acomposition for treatment of Gaucher disease comprising atherapeutically effective amount of a compound listed in Table 1,together with a pharmaceutically acceptable excipient. The administeredcompound may optionally comprise a salt of a compound of Table 1,particularly a pharmaceutically acceptable salt. In an embodiment of thetreatment method, the individual also suffers from Parkinson's disease.

In any of the aforesaid methods and compositions, the therapeuticcompound may comprise a beta-glucocerebrosidase inhibitor thatdemonstrates beta-glucocerebrosidase pharmacological chaperone activity.

In another embodiment, the invention includes a method of inducing astable conformation of a glucocerebrosidase, the method comprisingcontacting the glucocerebrosidase with ambroxol, or a derivativethereof. The invention also relates to a composition for inducing astable conformation of a glucocerebrosidase comprising ambroxol, or aderivative thereof. In some embodiments of the composition and methodfor inducing a stable conformation of a glucocerebrosidase, the ambroxolderivative comprises a cyclohexane ring, an amine group, and/or at leasttwo bromine groups. In some embodiments, the ambroxol derivativecomprises a compound listed in Table 2. In some embodiments, thederivate is an enantiomer, analog, ester, amide, prodrug, or metaboliteof ambroxol, or a salt of ambroxol, particularly a pharmaceuticallyacceptable salt. In some embodiments, the ambroxol derivative isbromhexine or a salt of bromhexine. In some embodiments, the salt ofambroxol or bromhexine is a hydrochloride.

In further embodiments, the invention includes (i) a method of inducinga stable conformation of a glucocerebrosidase, the method comprisingcontacting the glucocerebrosidase with a compound of Table 1, and (ii) acomposition for inducing a stable conformation of a glucocerebrosidasecomprising a compound of Table 1. The compound may optionally comprise asalt of a compound of Table 1, particularly a pharmaceuticallyacceptable salt.

In one embodiment of the method for inducing a stable conformation of aglucocerebrosidase, the glucocerebrosidase is contacted with ambroxol orderivative thereof, or with a compound of Table 1, in vitro. In anotherembodiment, the contact of the glucocerebrosidase occurs in vivo.

In some embodiments of the method for inducing a stable conformation ofa glucocerebrosidase, the glucocerebrosidase is selected from the groupconsisting of a wild type glucocerebrosidase, a mutantglucocerebrosidase, and an engineered glucocerebrosidase.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the present invention will now be described, by way ofexample only, with reference to the attached Figures.

FIG. 1 shows a heat denaturation curve of GCase in the presence ofambroxol. These data illustrate that increasing concentrations ofambroxol attenuate thermal denaturation of GCase.

FIG. 2 shows the activity of GCase (CEREZYME) in the presence ofincreasing concentrations of ambroxol. An IC50 of 27 μM was determinedin this assay.

FIG. 3 shows the activity of GCase (CEREZYME) at several different pHvalues, in the presence of ambroxol. The data illustrates that 25 μMambroxol only marginally inhibited GCase under acidic conditions.

FIGS. 4 a and 4 b show that ambroxol (abbreviated as ABX) enhanced boththe activity and concentration of wild-type GCase in fibroblasts thatwere pre-treated with ambroxol. FIG. 4 a shows the GCase activitydetermined by a fluorometric assay and FIG. 4 b shows the GCase proteinlevels as determined by densitometry.

FIGS. 5 a and 5 b show that ambroxol (abbreviated as ABX) enhanced boththe activity and protein levels of mutant GCase in Gaucher diseasefibroblast cell lines harboring either the N370S/N370S or F213I/L444Psets of missense mutations commonly associated with the most frequentlyoccurring type I form of Gaucher disease. FIG. 5 a shows the GCaseactivity determined by a fluorometric assay and FIG. 5 b shows the GCaseprotein levels as determined by densitometry.

FIGS. 6A-6I show the rescue of mutant GCase trafficking by ambroxol(abbreviated as ABX) compared to isofagomine (abbreviated as IFG) anduntreated controls (“Mock”). Immunohistochemical fluorescence labelingof GD fibroblast cell lines harboring the N370S/N370S missense mutationwere performed with rabbit polyclonal anti-GCase and mouse anti-Lamp-1(lysosomal membrane associated protein). Cell nuclear staining wasperformed with 4′-6′-diamidino-2-phenylindole. Colocalization of GCaseand Lamp-1 occurs at the lysosome in the presence of ambroxol (“ABX”),and to a lesser degree in the presence of isofagomine (“IFG”). (FIG. 6A,staining with anti-GCase in the presence of ABX; FIG. 6B, staining withanti-Lamp-1 in the presence of ABX; FIG. 6C, merger of FIGS. 6A and 6B;FIG. 6D, staining with anti-GCase in the presence of IFG; FIG. 6E,staining with anti-Lamp-1 in the presence of IFG; FIG. 6F, merger ofFIGS. 6D and 6E; FIG. 6G, staining with anti-GCase in the absence ofdrug; FIG. 6H, staining with anti-Lamp-1 in the absence of drug; FIG.6I, merger of FIGS. 6G and 6H.)

ABBREVIATIONS AND SHORT FORMS

The following abbreviations and short forms are used in thisspecification:

-   -   “ABX” refers to ambroxol.    -   “GCase” refers to beta-glucocerebrosidase.    -   “PC” refers to a pharmacological chaperone.    -   “IFG” refers to isofagomine.

DETAILED DESCRIPTION I. Definitions

As used in the specification and the appended claims, the singular forms“a,” “an” and “the” include plural referents unless the context clearlydictates otherwise.

The expressions “treat” and “treatment” mean cause, or the act ofcausing, a postponement of development of a disorder and/or a reductionin the severity of symptoms that will or are expected to develop. Theterms further include ameliorating existing symptoms, preventingadditional symptoms, and ameliorating or preventing the underlyingmetabolic causes of symptoms.

The expression “effective amount”, when used to describe therapy to anindividual, refers to the amount of a compound that results in atherapeutically useful effect.

As used herein, “individual” (as in the subject of the treatment) meansmammals, particularly non-human primates, e.g. apes and monkeys, andmost particularly humans.

The term “isolated compound” means a compound substantially free ofcontaminants or cell components with which the compound naturally occur,or the reagents used in synthesis or the byproducts of synthesis.“Isolated” and “substantially free of contaminants” does not mean thatthe preparation is technically pure (homogeneous), but it issufficiently pure to provide the compound in a form in which it can beused therapeutically.

A “derivative” compound, as the term is used herein, refers to a secondcompound that is derived from a first compound, such as a brominatedversion of a non-brominated parent compound. For example, bromhexine isa derivative of ambroxol.

II. Compounds of the Invention

Current therapies for Gaucher therapies are either expensive, or treatthe symptoms, but not the underlying cause of the disease. In contrast,the present invention describes compounds for use as “pharmacologicalchaperones”, and presents an opportunity to treat patients at a reducedcosts and with increased specificity.

Ambroxol, derivatives of ambroxol (e.g., bromhexine), and salts thereofare pharmacological chaperones (PC's), and as set forth in detailelsewhere herein, enhance the amounts of mutant enzyme that can betransported to and/or accumulated in the lysosome. Representativederivatives of ambroxol are set forth in Table 2, below. These compoundsmay be used as therapeutics for lysosomal storage disorders that arecharacterized by a mutant enzyme having reduced residual catalyticactivity in the lysosome due to retention of the mutant enzyme in theendoplasmic reticulum. Other compounds useful as therapeutics forlysosomal storage disorders that are characterized by a mutant enzymehaving reduced residual catalytic activity in the lysosome due toretention of the mutant enzyme in the endoplasmic reticulum includederivatives of ambroxol, as well as compounds that are GCase inhibitorshaving GCase pharmacological chaperone activity, set forth in Table 1,below.

Ambroxol, also known by its chemical nametrans-4-(2-amino-3,5-dibromobenzylamino)cyclohexanol, has the structure:

An aspect of the invention also includes a medicament, for the treatmentof Gaucher disease, comprising a pharmaceutically acceptable salt ofambroxol. In one embodiment, the salt is a hydrochloride salt.

Bromhexine, also known by its chemical name2-amino-3,5-dibromo-N-cyclohexyl-N-methylbenzenemethanamine, has thestructure:

An aspect of the invention also includes a medicament, for the treatmentof Gaucher disease, comprising a pharmaceutically acceptable salt ofbromhexine. In one embodiment, the salt is a hydrochloride salt.

Another aspect of the invention includes a pharmaceutically acceptablesalt of a derivative of ambroxol.

Yet another aspect of the invention includes a medicament, for thetreatment of Gaucher disease, comprising a compound having inhibitoryactivity against GCase. Table 1 lists compounds identified as GCaseinhibitors, which are believed to act as pharmacological chaperones.

TABLE 1 Beta-glucocerebrosidase inhibitors in GenPlus NINDS collectionCompound Name Structure CHLORPROMAZINE

DIENESTROL

DIFLUNISAL

FLUMETHAZONE PIVALATE

GENTIAN VIOLET

GUANABENZ ACETATE

MAPROTILINE HYDROCHLORIDE

MECLIZINE HYDROCHLORIDE

GLUCONOLACTONE

HALCINONIDE

PARAROSANILINE PAMOATE

ENOXACIN

CINNARAZINE

PERHEXILINE MALEATE

AMBROXOL HYDROCHLORIDE

ECONAZOLE NITRATE

SULCONAZOLE NITRATE

CLOMIPRAMINE HYDROCHLORIDE

ASTEMIZOLE

HOMIDIUM BROMIDE

SELAMECTIN

HYDROXYTACRINE MALEATE

THIORIDAZINE HYDROCHLORIDE

PYRVINIUM PAMOATE

PHENOLPHTHALEIN

NOSCAPINE HYDROCHLORIDE

PHENAZOPYRIDINE HYDROCHLORIDE

RESERPINE

TAMOXIFEN CITRATE

PERPHENAZINE

LANSOPRAZOLE

AMOXEPINE

PHENOXYBENZAMINE HYDROCHLORIDE

CLOPERASTINE HYDROCHLORIDE

THYROXINE

NARINGENIN

PIMOZIDE

NICARDIPINE HYDROCHLORIDE

N,N-HEXAMETHYLENEAMILORIDE

2,3-DIHYDROXY-6,7-DICHLOROQUINOXALINE

MIGLITOL

RITANSERIN

DIMERCAPTOPROPANOL

HYCANTHONE

APIGENIN

Table 2, below, illustrates structures of various Ambroxol derivativesand variants, together with the IC₅₀ values (in micromolar, resultsobtained from an assay using 0.3 mM MUbGlc as the substrate.

As can be seen in Table 2, compounds such as tribromophenyl propanoateare less effective inhibitors of GCase, whereas compounds comprising asecondary hydrophobic group in the form of a more flexible cyclohexanering are good inhibitors. Bromhexine shows a two fold reduction ininhibitory activity compared with ambroxol. Dibromophenyl derivativesbearing a alkyl amine with a flexible hydrophobic ring moiety are thuscandidates for possessing inhibitory activity against GCase. Therefore,another aspect of the invention includes a derivative of ambroxol,wherein the derivative comprises an alkyl amine with a flexiblehydrophobic ring moiety.

TABLE 2 Ambroxol derivatives demonstrating activity against humanbeta-glucocerebrosidase Compound (IC₅₀, micromolar) Structure Ambroxol(27)

Tribromophenyl Propanoate (>1000)

Dibromophenyl Benzamide (>1000)

Bromehexine (60)

Dibromoaniline (NI -1000)

Tribromoaniline (NI -1000)

a. Preparation of Compounds of the Invention

The compounds of the invention are known and may be prepared by methodsknown to the person skilled in the art of organic synthesis. Forexample, U.S. Patent Application publication number US2004/0242700,incorporated herein by reference in its entirety, provides a syntheticprotocol for the preparation of ambroxol.

b. Salts of Compounds of the Invention

For compounds that typically contain acidic or basic groups (such asamine or carboxyl groups) such groups will not necessarily be in thefree base form. When referring to compounds of the invention, thereference is intended to include salt forms of the compound. Within thescope of the invention, therefore, are salts of the active agent,especially salts of ambroxol and bromhexine. The preferred salts arepharmaceutically-acceptable salts. Also within the scope of theinvention are salts of derivatives of ambroxol.

The term “salts” embraces addition salts of free acids or free bases.The term “pharmaceutically-acceptable salt” refers to salts whichpossess toxicity profiles within a range that affords utility inpharmaceutical applications. Pharmaceutically unacceptable salts maynonetheless possess properties such as high crystallinity, which haveutility in the practice of the present invention, such as for exampleutility in process of synthesis, purification or formulation oftherapeutic compounds.

Suitable pharmaceutically-acceptable acid addition salts may be preparedfrom an inorganic acid or from an organic acid. Examples of inorganicacids include hydrochloric, hydrobromic, hydriodic, nitric, carbonic,sulfuric, and phosphoric acids. Appropriate organic acids may beselected from aliphatic, cycloaliphatic, aromatic, araliphatic,heterocyclic, carboxylic and sulfonic classes of organic acids, examplesof which include formic, acetic, propionic, succinic, glycolic,gluconic, lactic, malic, tartaric, citric, ascorbic, glucuronic, maleic,fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic,4-hydroxybenzoic, phenylacetic, mandelic, embonic (pamoic),methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic,trifluoromethanesulfonic, 2-hydroxyethanesulfonic, p-toluenesulfonic,sulfanilic, cyclohexylaminosulfonic, stearic, alginic, β-hydroxybutyric,salicylic, galactaric, oxalic, malonic and galacturonic acid. Examplesof pharmaceutically unacceptable acid addition salts include, forexample, perchlorates and tetrafluoroborates. All of these acid additionsalts may be prepared from ambroxol or bromhexine by reacting, forexample, the appropriate acid with the compound.

Suitable pharmaceutically acceptable base addition salts of ambroxolinclude, for example, metallic salts including alkali metal, alkalineearth metal and transition metal salts such as, for example, calcium,magnesium, potassium, sodium and zinc salts. Pharmaceutically acceptablebase addition salts also include organic salts made from basic aminessuch as, for example, N,N′-dibenzylethylenediamine, chloroprocaine,choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine)and procaine. Examples of pharmaceutically unacceptable base additionsalts include lithium salts and cyanate salts. All of these baseaddition salts may be prepared from ambroxol by reacting, for example,the appropriate base with the compound.

c. Pharmaceutical Compositions

In an aspect, the invention includes a composition comprising atherapeutically effective amount of ambroxol, derivatives of ambroxol,such as bromhexine, or a pharmaceutically acceptable salt thereof, inconjunction with a pharmaceutically acceptable excipient for treatmentof an individual suffering from Gaucher disease. In another aspect, theinvention includes a composition comprising a therapeutically effectiveamount of ambroxol or a derivative thereof, inclusive ofpharmaceutically acceptable salts thereof, in conjunction with apharmaceutically acceptable excipient for treatment of Gaucher diseasein an individual suffering from both Gaucher disease and Parkinson'sdisease. In yet another aspect, the invention includes a compositioncomprising a therapeutically effective amount of a derivative ofambroxol, including, but not limited to an enantiomer, analog, ester,amide, prodrug, or metabolite of ambroxol, or a pharmaceuticallyacceptable salt thereof, in conjunction with a pharmaceuticallyacceptable excipient for treatment of an individual suffering fromGaucher disease. Reports of Parkinson's disease associated with type IGaucher's disease have been documented, suggesting a genetic linkbetween the two diseases (Intern Med. (2006) 45(20):1165-1167).

In another aspect, the invention includes a composition comprising atherapeutically effective amount of a GCase inhibitor from Table 2 thatdemonstrates GCase PC activity, or a pharmaceutically acceptable saltthereof, in conjunction with a pharmaceutically acceptable excipient fortreatment of Gaucher disease in an individual suffering from Gaucherdisease. In yet another aspect, the invention includes a compositioncomprising a therapeutically effective amount of a GCase inhibitor fromTable 2 that demonstrates GCase PC activity, or a pharmaceuticallyacceptable salt thereof in conjunction with a pharmaceuticallyacceptable excipient for treatment of Gaucher disease in an individualsuffering from both Gaucher disease and Parkinson's disease.

The active agent (e.g., ambroxol, derivatives of ambroxol, compounds ofTable 1, or pharmaceutically acceptable salts thereof) may beadministered in the form of a pharmaceutical composition, in combinationwith a pharmaceutically acceptable carrier. The active ingredient insuch formulations may comprise from 0.1 to 99.99 weight percent.“Pharmaceutically acceptable carrier” means any carrier, diluent orexcipient which is compatible with the other ingredients of theformulation and not deleterious to the recipient.

The active agent is preferably administered with a pharmaceuticallyacceptable carrier selected on the basis of the selected route ofadministration and standard pharmaceutical practice. The active agentmay be formulated into dosage forms according to standard practices inthe field of pharmaceutical preparations. See Alphonso Gennaro, ed.,Remington's Pharmaceutical Sciences, 18th Edition (1990), MackPublishing Co., Easton, Pa. Suitable dosage forms may comprise, forexample, tablets, capsules, solutions, parenteral solutions, troches,suppositories, or suspensions. For examples of the preparation of oral,topical, suppository and parenteral formulations of ambroxol, alsouseful for administration of bromhexine, or other ambroxol derivatives,or for a GCase inhibitor that demonstrates GCase PC activity, seeExamples 1-8 of WO2005/007146, or its equivalent US2005/00148747,incorporated herein by reference.

In another embodiment, ambroxol, a derivative of ambroxol, or a GCaseinhibitor that demonstrates GCase PC activity, is used in thepreparation of a medicament for the treatment of Gaucher disease.

For parenteral administration, the active agent may be mixed with asuitable carrier or diluent such as water, an oil (particularly avegetable oil), ethanol, saline solution, aqueous dextrose (glucose) andrelated sugar solutions, glycerol, or a glycol such as propylene glycolor polyethylene glycol. Solutions for parenteral administrationpreferably contain a water soluble salt of the active agent. Stabilizingagents, antioxidant agents and preservatives may also be added. Suitableantioxidant agents include sulfite, ascorbic acid, citric acid and itssalts, and sodium EDTA. Suitable preservatives include benzalkoniumchloride, methyl- or propyl-paraben, and chlorbutanol. The compositionfor parenteral administration may take the form of an aqueous ornon-aqueous solution, dispersion, suspension or emulsion.

For oral administration, the active agent may be combined with one ormore solid inactive ingredients for the preparation of tablets,capsules, pills, powders, granules or other suitable oral dosage forms.For example, the active agent may be combined with at least oneexcipient such as fillers, binders, humectants, disintegrating agents,solution retarders, absorption accelerators, wetting agents absorbentsor lubricating agents. According to one tablet embodiment, the activeagent may be combined with carboxymethylcellulose calcium, magnesiumstearate, mannitol and starch, and then formed into tablets byconventional tableting methods.

The compositions are preferably formulated in a unit dosage form, eachdosage containing from about 50 to about 1000 mg, more typically, about250 to about 500 mg of active agent per unit dosage. The term “unitdosage form” refers to physically discrete units suitable as a unitarydosage for human subjects and other mammals, each unit containing apredetermined quantity of active material calculated to produce thedesired therapeutic effect, in association with a suitablepharmaceutical excipient.

The pharmaceutical compositions of the present invention may also beformulated so as to provide slow or controlled release of the activeingredient therein using, for example, hydropropylmethyl cellulose invarying proportions to provide the desired release profile, otherpolymer matrices, gels, permeable membranes, osmotic systems, multilayercoatings, microparticles, liposomes and/or microspheres.

In general, a controlled-release preparation is a pharmaceuticalcomposition capable of releasing the active ingredient at the requiredrate to maintain constant pharmacological activity for a desirableperiod of time. Such dosage forms provide a supply of a drug to the bodyduring a predetermined period of time and thus maintain drug levels inthe therapeutic range for longer periods of time than conventionalnon-controlled formulations.

U.S. Pat. No. 5,674,533 discloses controlled-release pharmaceuticalcompositions in liquid dosage forms for the administration ofmoguisteine, a potent peripheral antitussive. U.S. Pat. No. 5,059,595describes the controlled-release of active agents by the use of agastro-resistant tablet for the therapy of organic mental disturbances.U.S. Pat. No. 5,591,767 describes a liquid reservoir transdermal patchfor the controlled administration of ketorolac, a non-steroidalanti-inflammatory agent with potent analgesic properties. U.S. Pat. No.5,120,548 discloses a controlled-release drug delivery device comprisedof swellable polymers. U.S. Pat. No. 5,073,543 describescontrolled-release formulations containing a trophic factor entrapped bya ganglioside-liposome vehicle. U.S. Pat. No. 5,639,476 discloses astable solid controlled-release formulation having a coating derivedfrom an aqueous dispersion of a hydrophobic acrylic polymer.Biodegradable microparticles are known for use in controlled-releaseformulations. U.S. Pat. No. 5,354,566 discloses a controlled-releasepowder that contains the active ingredient. U.S. Pat. No. 5,733,566,describes the use of polymeric microparticles that release antiparasiticcompositions.

The controlled-release of the active ingredient may be stimulated byvarious inducers, for example pH, temperature, enzymes, water, or otherphysiological conditions or compounds. Various mechanisms of drugrelease exist. For example, in one embodiment, the controlled-releasecomponent may swell and form porous openings large enough to release theactive ingredient after administration to a patient. The term“controlled-release component” in the context of the present inventionis defined herein as a compound or compounds, such as polymers, polymermatrices, gels, permeable membranes, liposomes and/or microspheres, thatfacilitate the controlled-release of the active ingredient in thepharmaceutical composition. In another embodiment, thecontrolled-release component is biodegradable, induced by exposure tothe aqueous environment, pH, temperature, or enzymes in the body. Inanother embodiment, sol-gels may be used, wherein the active ingredientis incorporated into a sol-gel matrix that is a solid at roomtemperature. This matrix is implanted into a patient, preferably amammal, having a body temperature high enough to induce gel formation ofthe sol-gel matrix, thereby releasing the active ingredient into thepatient.

Compositions of the compounds of the invention that are suitable foradministration intranasally or by inhalation are of particular interest.

The compounds of the invention can be administered intranasally or byinhalation, typically in the form of a dry powder (either alone, as amixture, for example, in a dry blend with lactose in anhydrous ormonohydrate form, preferably monohydrate, mannitol, dextran, glucose,maltose, sorbitol, xylitol, fructose, sucrose or trehalose, or as amixed component particle, for example, mixed with phospholipids) from adry powder inhaler or as an aerosol spray from a pressurized container,pump, spray, atomizer (preferably an atomizer using electrohydrodynamicsto produce a fine mist), or nebulae, with or without the use of asuitable propellant, such as dichlorofluoromethane.

The pressurized container, pump, spray, atomizer, or nebulae contains asolution or suspension of the active compound comprising, for example,ethanol (optionally, aqueous ethanol) or a suitable alternative agentfor dispersing, solubilizing, or extending release of the active, thepropellant(s) as solvent and an optional surfactant, such as sorbitantrioleate or an oligolactic acid.

Prior to use in a dry powder or suspension formulation, the drug productis micronized to a size suitable for delivery by inhalation (typicallyless than 5 microns). This may be achieved by any appropriatecomminuting method, such as spiral jet milling, fluid bed jet milling,supercritical fluid processing to form nanoparticles, high pressurehomogenization, or spray drying.

A suitable solution formulation for use in an atomizer usingelectrohydrodynamics to produce a fine mist may contain from 1 μg to 20mg of the compound of the invention per actuation and the actuationvolume may vary from 1 μL to 100 μL. A typical formulation may comprisethe compound of the invention, propylene glycol, sterile water, ethanoland sodium chloride. Alternative solvents which may be used instead ofpropylene glycol include glycerol and polyethylene glycol.

Capsules, blisters and cartridges (made, for example, from gelatin orHPMC) for use in an inhaler or insufflator may be formulated to containa powder mix of the nicotinamide derivative of formula (I), a suitablepowder base such as lactose or starch and a performance modifier such asL-leucine, mannitol, or magnesium stearate.

Formulations for inhaled/intranasal administration may be formulated tobe immediate and/or modified release. Modified release formulationsinclude delayed-, sustained-, pulsed-, controlled dual-, targeted- andprogrammed-release formulations. Sustained or controlled release can beobtained by using, for example, poly(D,L-lactic-co-glycolic acid).

III. Activity of the Compounds of the Invention

A functional compound which is able to induce a stable molecularconformation of an enzyme, such as beta-glucocerebrosidase (GCase), canserve as a “pharmacological chaperone” (PC) for the enzyme bystabilizing the enzyme in a proper conformation for transport to thelysosome. In one aspect, the compound induces a stable molecularconformation of a wild type enzyme for transport to the lysosome. In anaspect, the compound induces a stable molecular conformation of a mutantenzyme for transport to the lysosome. In another aspect, the compoundinduces a stable molecular conformation of an engineered enzyme fortransport to the lysosome.

A “wild type enzyme,” as used herein, refers to an enzyme that has theamino acid sequence of the enzyme as encoded by the gene for the enzyme.An “engineered” enzyme, as the term is used herein, refers to an enzymethat has been modified through one or more techniques such as, but notlimited to, recombinant protein production, site-directed mutagenesis,chemical modification, in vitro glycosylation, and in vivoglycosylation. A “mutant” enzyme, as used herein, refers to an enzymethat has an amino acid sequence that differs from the wild type sequenceby one or more amino acids. A mutant enzyme may have the same number ofamino acids as the wild type enzyme, or the mutant enzyme may have atleast one more or at least one fewer amino acid residue than the wildtype enzyme. A mutant enzyme may occur naturally or may be engineered,as described herein.

In an embodiment of the invention, an effective amount of compound isused to induce the stable conformation. Some inhibitors of the enzymeare known to occupy the catalytic center of the enzyme, resulting instabilization of its conformation in vitro. The inhibitors may bind tothe native enzyme, the wild type enzyme, or to recombinantly-producedenzyme obtained from any source, including, but not limited to bacterialcells, insect cells, plant cells and mammalian cells. Other compoundsmay bind close to, but not on, the active site. Yet, other compounds maybind at a distance from the active site while stabilizing the enzyme asallosteric pharmacological chaperones. All such compounds may also serveas PC's to enforce the proper folding of the enzyme in vivo, and thusrescue the mutant enzyme from degradation by the endoplasmic reticulumquality control system.

In an aspect of the invention, ambroxol, a derivative of ambroxol, or aGCase inhibitor that demonstrates GCase PC activity, such as a compoundof Table 1, or pharmaceutically acceptable salt thereof, has theproperty of functioning as a chaperone, also referred to herein as a PC,for GCase. As used herein, “pharmacological chaperone” means that acompound of the invention has the ability to facilitate and/or enhancethe transport of a biological molecule across a membrane or into asubcellular compartment. In particular, a pharmacological chaperone ofthe invention is a compound that has the ability to facilitate orenhance the transport of GCase into the lysosome. In another aspect, theaforementioned compounds demonstrate inhibitory activity with respect toGCase.

A PC can stabilize its target enzyme (e.g., ambroxol stabilization ofGCase or mutant GCase) into a stable conformation and thus allow theenzyme to successfully avoid the endoplasmic reticulum-associateddegradation system. The ability of the compounds to bind to GCase andfacilitate the transport of GCase into the lysosome is assayed using aGCase assay as set forth in detail in the Example below. In anembodiment of the invention, a compound binding and/or GCase chaperoneassay is conducted by assaying GCase activity in isolated fibroblastcells.

While not wishing to be bound by any particular theory or mechanism ofaction, ambroxol and derivatives thereof function as specific molecularguides for a mutant GCase and enhance GCase activity in the lysosome. Ithas been found that at least ambroxol binds to mutant GCase with a lowaffinity at the pH of the endoplasmic reticulum, thereby promotingproper folding of GCase. The enhancement of proper folding enhances thechaperoning of GCase to the lysosome by ambroxol. In the lysosome,ambroxol dissociates from GCase due to the very low affinity of ambroxolfor GCase at lysosomal pH. Furthermore, because of the highconcentration of accumulated glucocerebrosides in the lysosome, theglucocerebrosides, which are natural substrates of GCase, shift thebinding equilibrium to displace ambroxol from GCase.

IV. Methods of Treatment Using Compounds of the Invention

Ambroxol, a derivative of ambroxol, or a GCase inhibitor of Table 1 thatdemonstrates GCase PC activity, or pharmaceutically acceptable saltsthereof, are used for the treatment of Gaucher disease, which canbenefit from increased GCase activity in the lysosome. The increasedtransport of GCase into the lysosome, and thereby, the increasedconcentration of GCase in the lysosome, will increase the GCase activityin the lysosome. Even mutant forms of GCase, such as the mutant GCaseforms characteristic of Gaucher disease, which have low levels of GCaseactivity when compared with wild type (i.e., non-mutated) GCase, canincrease GCase activity in the lysosome when transported to thelysosome.

Thus, in one aspect, a compound as described above is administered totreat Gaucher disease. The method comprises administering an effectiveamount of the compound, or a pharmaceutical composition comprising sucha compound, to an individual in need of such treatment.

Gaucher disease is a condition in which a patient suffers from anaccumulation of glycosphingolipids. This disease arises because of adeficiency in the GCase enzyme responsible for the catabolism of theglycosphingolipids. An increase in the GCase activity in the lysosomesof patients afflicted with Gaucher disease can treat the disease byenhancing the catabolism of the accumulating glycosphingolipids.Therefore, an individual who is in need of treatment with a compoundaccording to the invention can be an individual who is suffering fromone or more symptoms of Gaucher disease.

In an aspect, the patient treated for Gaucher's disease is characterizedby a mutation in the gene encoding GCase, wherein the resulting enzymemutation is selected from the group consisting of V15L, G46E, K79N,R119Q, P122S, R131L, K157Q, N188S, Y212H, F213I, F216V, F216Y, F251L,R257E, P289L, A309V, H311R, W312C, Y323I, G325R, C342G, R353G, R359X(termination), S364T, N370S, L371V, G377S, V394L, V398F, P401L, D409H,D409V, P415R, L444P, R463C, G478S, R496H. In another aspect, the patientis characterized by more than one mutation in the gene encoding GCase,selected from the group consisting of (L444P, A456P, V460V), (D140H,E326K), and (H255Q, D409H). In yet another aspect, the patient ischaracterized by an insertion, deletion, truncation, or frameshiftmutation, or combination thereof, in the gene encoding the GCase,selected from the group consisting of:

-   -   84GG (guanine insertion),    -   splice site mutation in intron 2 (IVS2DS+1G-A), resulting in the        skipping of exon 2,    -   a 1-bp deletion (1023delC in the genomic sequence) in the GCase        gene,    -   a 55-bp deletion (nucleotides 5879-5933 in genomic DNA) in the        GCase gene,    -   a homozygous 259C-T transition (1763 in the genomic DNA)    -   a homozygous 1-bp deletion in the GCase gene, resulting in a        frameshift and premature truncation of the protein in exon 6,        and    -   G-to-A substitution at the first position in the splice site of        intron 10 of the GCase gene, resulting in the insertion of the        first 11 basepairs of IVS10 and deletion of the first 11        basepairs of exon 11.

In one aspect, the patient treated for Gaucher's disease ischaracterized by a mutation in the gene encoding GCase, wherein theresulting enzyme mutation is selected from the group consisting of N370Sand L444P.

In another aspect, a compound of the invention is used to treat Gaucherdisease in a patient having both Gaucher disease and Parkinson'sDisease. The method comprises administering an effective amount of acompound as described above, or a pharmaceutical composition comprisingthe compound, as described herein, to an individual in need of suchtreatment.

In one embodiment, a method of the invention includes treating anindividual suffering from Gaucher disease comprising administering tothe individual an effective amount of ambroxol or derivative thereof. Inanother embodiment, a method of the invention includes treating anindividual suffering from both Gaucher disease and Parkinson's diseasecomprising administering to the individual an effective amount ofambroxol or derivative thereof. In yet another embodiment, a method ofthe invention includes treating an individual suffering from Gaucherdisease comprising administering to the individual an effective amountof a derivative of ambroxol or derivative thereof, or a GCase inhibitorthat demonstrates GCase PC activity. In another embodiment, a method ofthe invention includes treating an individual suffering from bothGaucher disease and Parkinson's disease comprising administering to theindividual an effective amount of ambroxol or derivative thereof, or aGCase inhibitor that demonstrates GCase PC activity. In each of theaforesaid embodiments, the administered compound may comprise a salt,particularly a pharmaceutically acceptable salt. In some embodiments,the salt is a hydrochloride salt.

The amount of the therapeutic compound of the invention that will beeffective in the treatment of a particular disorder or condition willdepend on the nature of the disorder or condition, and is determined bystandard clinical techniques. In addition, in vitro assays mayoptionally be employed to help identify optimal dosage ranges. Theprecise dose to be employed in the formulation also will depend on theroute of administration and the seriousness of the disease, disorder, orcondition and is decided according to the judgment of the practitionerand each patient's circumstances.

V. Administration of Compounds of the Invention

In a preferred embodiment, the compounds of the invention areadministered orally to a patient. However, the compounds may beadministered by any route, including by rectal, pulmonary, sublingual,and parenteral administration. Parenteral administration includes, forexample, intravenous, intramuscular, intraarterial, intraperitoneal,intranasal, intravaginal, intravesical (e.g., to the bladder),intradermal, transdermal, topical or subcutaneous administration.

Typically it is contemplated that treatment would be given in a dosesufficient to achieve concentrations where the chaperone binds to andstabilizes GCase and in a dosing interval long enough to effect lowconcentrations of the chaperone and dissociation from the enzyme. Theseoptimal pharmacokinetic properties depend on he bioavalability and theelimination half-life of the chaperone. The dosing interval may be oncea week, twice a week, every-other-day, once per day, typically once,twice, three times or four times per day with the doses given at equalintervals throughout the day and night in order to maintain a constantpresence of the drug in order to induce sufficient transport of GCaseinto the lysosome. However, the skilled artisan will be aware that atreatment schedule can be optimized for any given patient, and thatadministration of compound may occur less frequently than once per day.

One or more compounds of the invention may be administeredsimultaneously, by the same or different routes, or at different timesduring treatment. The compounds of the invention may also be prescribedto be taken in combination with other drugs used to treat Gaucherdisease and/or Parkinson's disease. When used in such combinationscompounds of the invention and conventional drugs may be administeredsimultaneously, by the same or different routes, or at different timesduring treatment. The dose of the conventional drug selected will dependon the particular compound being used and the route and frequency ofadministration.

The treatment may be carried out for as long a period as necessary.Typically it is contemplated that treatment would be continuedindefinitely while the disease state persists, although discontinuationmight be indicated if the compounds no longer produce a beneficialeffect. The treating physician will know how to increase, decrease, orinterrupt treatment based on patient response.

The specific dose of a compound according to the invention to obtaintherapeutic benefit for treatment of Gaucher disease will, of course, bedetermined by the particular circumstances of the individual patientincluding the size, weight, age and sex of the patient, the nature andstage of the disease, the aggressiveness of the disease, and the routeof administration of the compound.

For example, a daily dosage from about 0.02 to about 50 mg/kg/day may beutilized, more preferably from about 0.1 to about 10 mg/kg/day. Higheror lower doses are also contemplated as it may be necessary to usedosages outside these ranges in some cases. The daily dosage may bedivided, such as being divided equally into two to four times per daydaily dosing. Suitable dosage ranges for intravenous administration aregenerally about 20-500 micrograms of active compound per kilogram bodyweight.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Optionally associated withsuch container(s) is a notice in the form prescribed by a governmentalagency regulating the manufacture, use or sale of pharmaceuticals orbiological products, which notice reflects approval by the agency ofmanufacture, use or sale for human administration.

In accordance with the present invention, as described above or asdiscussed in the Example below, there can be employed conventionalclinical, chemical, cellular, histochemical, biochemical, molecularbiology, microbiology and recombinant DNA techniques which are known tothose of skill in the art. Such techniques are explained fully in theliterature.

Without further description, it is believed that one of ordinary skillin the art can, using the preceding description and the followingillustrative example, make and utilize the compounds of the presentinvention and practice the claimed methods. The following workingexample therefore, specifically points out the preferred embodiments ofthe present invention, and is not to be construed as limiting in any waythe remainder of the disclosure.

Example Chaperoning of Wild Type Beta-Glucocerebrosidase and GaucherDisease Mutant Beta-glucocerebrosidases by Compounds of the Invention A.Assay Conditions for GCase Activity and Function Studies.

Experiments set forth herein and related to the activity and function ofGCase in the presence and absence of compounds of the invention wereconducted using imiglucerase (CEREZYME, Genzyme) as the GCase. Cellularmaterials were obtained from fibroblast lysates expressing wild-typeGCase and separately from fibroblast lysates derived from Gaucherdisease patient cell lines carrying genotypes that encode either theN370S/N370S or F213I/L444P substitutions in GCase.

Quantitative determinations of enzymatic activity were performed by wayof fluorometric assays utilizing the artificial substrate4-methylumbelliferyl 2-acetamido-2-deoxy-beta-D-glucopyranoside (MUGlc).Reactions were performed at room temperature for 15-30 minutes, asindicated, using equal aliquots of enzyme (25 μL 20 mM Citrate PhosphatepH 5.5, 0.4% Taurodeoxycholate (TC)) and substrate (25 μL, 20 mM CitratePhosphate pH 5.5, MUGlc 1.6 mM). The amount of MU-fluorophore releasedin each assay was monitored fluorometrically using aspectrophotofluorometer following the addition of a 4-fold (200 μl)excess of 0.1M MAP, pH 10.5. The observed activity is either expressedin relative fluorescence units (FUs), as a percentage of the activityobserved in untreated GCase controls (% residual activity), or as thefold-increase as compared to lysates from untreated cells (relativeincrease in activity).

Confluent cultures of patient fibroblasts were grown in 24-well plates.Following a five day treatment of the fibroblasts with either DMSO ortest compound, the cells were washed twice with phosphate bufferedsaline, then lysed on ice for 30 minutes using 20 mM citrate-phosphatebuffer, pH 5.5, containing 0.4% TC. GCase activity was analyzed in a 25μl aliquot of the cell lysate using the same protocol described abovefor the purified enzyme.

B. Thermal Stability of GCase in the Presence of Ambroxol.

FIG. 1 shows a heat denaturation curve of GCase in the presence ofambroxol at concentrations of 3 μM, 6 μM, and 24 μM. Ambroxol was shownto attenuate thermal denaturation of wild type (WT) GCase, increasingthe half life at 50° C., more than three-fold at a concentration of 20μM. The data illustrate that increasing concentrations of ambroxolattenuate thermal denaturation of GCase, evidencing ambroxol binding toGCase.

GCase was also incubated in the presence of increasing concentrations ofambroxol or DMSO (control, compound solvent) at 50° C. for varyingperiods of time. Similar to samples maintained at 4° C., the fraction ofGCase activity remaining after incubation at 50° C. increased in samplescontaining increasing concentrations of ambroxol as compared to samplescontaining only DMSO. The increase in GCase activity in the presence ofthe highest concentration of ambroxol was more than three-fold.

C. Kinetic Analysis of GCase Activity in the Presence of Ambroxol andOther GCase Inhibitors.

FIG. 2 shows the activity of GCase in the presence of increasingconcentration of ambroxol. Kinetic analysis showed ambroxol to be acompetitive inhibitor of GCase with a K_(i) of 10 μM. The IC₅₀ forambroxol as an inhibitor of GCase was determined to be 27 μM (FIG. 2).The IC₅₀ for bromhexine was determined to be 60 μM.

An additional experiment was conducted to compare the activity ofambroxol with the activity of the known GCase inhibitor, isofagomine.Ambroxol and isofagomine both attenuated thermal denaturation of wildtype (WT) GCase, increasing the half life of GCase more than three-foldat a concentration of 20 μM, at 50° C. Significantly, ambroxol binds tomisfolded GCase with lower affinity than that of isofagomine at the pHof the endoplasmic reticulum, where ambroxol promotes proper GCasefolding, and thus, enhances GCase traffic to the lysosome. In thelysosome, ambroxol dissociates from GCase due to the very low affinityof ambroxol for GCase at lysosomal pH. Furthermore, the accumulatedglucocerobrosides (the natural substrates of GCase) in the lysosome alsoserve to displace ambroxol. While not wishing to be bound by anyparticular theory, ambroxol may be less likely to be trapped in thelysosome than isofagomine, and therefore, the risk that long-termtreatment with ambroxol will cause ambroxol to accumulate in thelysosome at a concentration sufficient to become a GCase inhibitor andparadoxically cause Gaucher disease is lower than for isofagomine.

The activity of GCase was also assayed at several different pH values,including 4.5, 5.0, 5.5, 6.0, 6.5 and 7.0. As a point of reference, thepH of the lysosome is acidic (pH<5.5) and the pH of the endoplasmicreticulum is near neutral. As shown in FIG. 3, ambroxol only marginallyinhibited GCase under acidic conditions.

This finding supports the role of ambroxol and derivatives thereof aspharmacological chaperones (PC's). In order for a PC to stabilize itstarget protein (e.g., ambroxol stabilization of GCase or mutant GCase)and thus allow the enzyme to successfully avoid the endoplasmicreticulum-associated degradation system, a PC should possess anappropriate level of binding-affinity at neutral pH. Followingenzyme-ligand complex formation at neutral pH and the subsequentsuccessful trafficking of the enzyme to its appropriate organelle (inthe present case, the acidic lysosome), a drop in affinity would beadvantageous to facilitate the displacement of the inhibitory PC by thenative substrate of the enzyme or the mutant enzyme.

The data set forth herein demonstrates that the affinity of ambroxol forGCase would be low to negligible following incorporation into the low-pHlysosome, and that the affinity of GCase for ambroxol would be maximalin the pH-neutral ER, the location where affinity is most desirable.Thus, the data set forth herein demonstrate that ambroxol is anexcellent therapeutic pharmacological chaperone for GCase.

D. Ambroxol as a Pharmacological Chaperone of GCase.

Fibroblasts expressing wild-type GCase were treated with ambroxol for 5days prior to measuring GCase activity levels within the cells. Ambroxol(abbreviated “ABX” in FIG. 4 a and FIG. 4 b) was found to enhance boththe GCase activity and the levels of wild-type GCase by more than oneand half times that of non-treated cells. FIG. 4 a shows the GCaseactivity determined by a fluorometric assay. FIG. 4 b shows the GCaseprotein levels as determined by densitometry.

Even greater increases of GCase activity and enzyme levels were observedin Gaucher disease patient-derived fibroblast cell lines harboring theGaucher disease associated genotypes N370S/N370S and F213I/L444P. FIGS.5 a and 5 b show that ambroxol (abbreviated as ABX) enhanced both theactivity and protein levels of mutant GCase in Gaucher diseasefibroblast cell lines harboring either the N370S/N370S or F213I/L444Psets of missense mutations. These mutations are commonly associated withthe most frequently occurring type I form of Gaucher disease. FIG. 5 ashows the GCase activity determined by a fluorometric assay. FIG. 5 bshows the GCase protein levels as determined by densitometry.

GCase activity and GCase concentration in the presence of 60 μM ambroxolwere approximately 3 and 2.5 times the activity and concentration ofnon-treated Gaucher disease patient fibroblasts, respectively. Type IGaucher disease patients have residual enzyme levels that areapproximately 10-15% of normal. The data set forth herein suggests that,similar to other lysosomal storage disorders, it appears that only arelatively small increase in GCase activity is necessary to treat theclinical progression of the disease.

Immunohistochemical fluorescence labeling of Gaucher disease fibroblastcell lines harboring the N370S/N370S missense mutation was performedwith rabbit polyclonal anti-GCase and mouse anti-Lamp-1 (lysosomalmembrane associated protein). Cell nuclear staining was performed with4′-6′-diamidino-2-phenylindole. Colocalization of GCase and Lamp-1 inthe presence of ambroxol is indicative of rescued trafficking andlysosomal delivery of GCase. Restored activity and trafficking of mutantGCase are the ultimate goals of a pharmacological chaperone-basedtherapeutic strategy. FIGS. 6A-6I shows the rescue of mutant GCasetrafficking by ambroxol (abbreviated as ABX). Colocalization of GCaseand Lamp-1 occurs at the lysosome in the presence of ambroxol (“ABX”;FIG. 6C), and to a lesser degree in the presence of isofagomine (“IFG”;FIG. 6F).

In summary, compounds set forth herein, including ambroxol andderivatives thereof, attenuate thermal denaturation of GCase and act aspharmacological chaperones for GCase into the lysosome. Accordingly,they are useful for the treatment of patients suffering from Gaucherdisease and also of Gaucher disease patients who may also be sufferingfrom Parkinson's disease.

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety.

While the invention has been disclosed with reference to specificembodiments, it is apparent that other embodiments and variations ofthis invention may be devised by others skilled in the art withoutdeparting from the true spirit and scope of the invention. The appendedclaims are intended to be construed to include all such embodiments andequivalent variations.

1. (canceled)
 2. A method of treating a lysosomal storage disordercomprising administering to a patient a pharmaceutically acceptableamount of ambroxol, ambroxol derivative or a pharmaceutically acceptablesalt thereof.
 3. The method of claim 2, comprising administeringambroxol or a pharmaceutically acceptable salt thereof.
 4. The method ofclaim 3, wherein said patient is also administered another drug for thetreatment of Gaucher's Disease.
 5. The method of claim 3, wherein saidpatient is also administered another drug for the treatment ofParkinson's Disease.
 6. The method of claim 3, wherein said patient hasGaucher's Disease.
 7. The method of claim 6, wherein said patient alsohas Parkinson's disease.
 8. The method of claim 6, wherein said patienthas a mutation in the gene encoding a beta glucocerebrosidase.
 9. Themethod of claim 2, wherein the ambroxol derivative is selected fromambroxol hydrochloride, tribromophenyl propanoate, dibromophenylbenzamide, bromhexine, dibromoanilin, or tribromoaniline or apharmaceutically acceptable salt thereof.
 10. The method of claim 9,wherein the ambroxol derivative is bromhexine or a pharmaceuticallyacceptable salt thereof.
 11. The method of claim 10, wherein saidpatient is also administered another drug for the treatment of Gaucher'sDisease.
 12. The method of claim 10, wherein said patient is alsoadministered another drug for the treatment of Parkinson's Disease. 13.The method of claim 10, wherein said patient has Gaucher's Disease. 14.The method of claim 13, wherein said patient also has Parkinson'sdisease.
 15. The method of claim 13, wherein said patient has a mutationin the gene encoding a beta glucocerebrosidase.
 16. The method of claim9, wherein the ambroxol derivative is ambroxol hydrochloride.
 17. Themethod of claim 16, wherein said patient is also administered anotherdrug for the treatment of Gaucher's Disease.
 18. The method of claim 16,wherein said patient is also administered another drug for the treatmentof Parkinson's Disease.
 19. The method of claim 16, wherein said patienthas Gaucher's Disease.
 20. The method of claim 19, wherein said patientalso has Parkinson's disease.
 21. The method of claim 19, wherein saidpatient has a mutation in the gene encoding a beta glucocerebrosidase.